PORPHYRINS AND BILE PIGMENTS

WHAT ARE PORPHYRINS?

Porphyrins are cyclic compounds formed by the linkage of 4 pyrrole rings through HC=methenyl bridges

 A characteristic property of the porphyrins is the formation of complexes with metal ions bound to the nitrogen atom of the pyrrole rings Examples are the iron porphyrins such as heme of hemoglobin and the magnesiumcontaining porphyrin chlorophyll, the photosynthetic pigment of plants.

WHAT IS HEME?
Heme is a prosthetic group that consists of an iron atom contained in the center of the porphyrin ring

 A hemoprotein or heme protein, is a metalloprotein containing a heme prosthetic group, either covalently or noncovalently bound to the protein itself The iron in the heme is capable of undergoing oxidation and reduction (usually to +2 and +3)

Examples of Some Important Human and Animal Hemoproteins

Protein Hemoglobin Myoglobin Cytochrome c Cytochrome P450 Catalase Tryptophan pyrrolase Function Transport of oxygen in blood Storage of oxygen in muscle Involvement in electron transport chain Hydroxylation of xenobiotics Degradation of hydrogen peroxide Oxidation of tryptophan

HEME NOMENCLATURE
The porphyrins found in nature are compounds in which various side chains are substituted for the eight hydrogen atoms numbered in the porphyrin nucleus

 As a simple means of showing these substitutions, Fischer proposed a shorthand formula in which the methlene bridges are omitted and each pyrrole ring is shown with the eight substituent positions

UROPORPHYRIN III

A (acetate) = CH2COOH P (propionate) = CH2CH2COOH Note the asymmetry of substituents in ring IV A porphyrin with this type of asymmetric substitution is classified as a type III porphyrin

TYPES OF PORPHYRINS

A porphyrin with a completely symmetric arrangement of the substituents is classified as a type I porphyrin. Only types I and III are found in nature, and the type III series is far more abundant

PROTOPORPHYRIN IX AND HEME

Heme and its immediate precursor, protoporphyrin IX, are both type III porphyrins However, they are sometimes identified as belonging to series IX, because they were designated ninth in a series of isomers postulated by Hans Fischer

HEME SYNTHESIS
mitochondria

cytosol

Starting materials: Succinyl-CoA and Glycine Condensation with the help of pyridoxal phosphate produces -amino--ketoadipate

HEME SYNTHESIS
mitochondria

cytosol

Decarboxylation produces ALA, catalyzed by the rate controlling enzyme in porphyrin biosynthesis, ALA synthase 2 ALA molecules condense to form porphobilinogen (PBG), catalyzed by ALA dehydratase ALA dehydratase is a zinc-containing enzyme and is inhibited by lead

HEME SYNTHESIS
4 PBG molecules condense to form hydroxymethylbilane (HMB), catalyzed by uroporphyrinogen I synthase (PBG deaminase/HMB synthase)

HEME SYNTHESIS
HMB cyclizes spontaneously to form uroporphyrinogen I or is converted to uroporphyrinogen III by the action of uroporphyrinogen III synthase

Also called PBG deaminase or HMB synthase.

HEME SYNTHESIS
Under normal conditions, the uroporphyrinogen formed is almost exclusively the III isomer In certain diseases (porphyrias), the type I isomers of porphyrinogens are formed in excess.

HEME SYNTHESIS
Uroporphyrinogens I and III have the pyrrole rings connected by methylene bridges (CH2), which do not form a conjugated ring system. Thus, these compounds are colorless

HEME SYNTHESIS
The porphyrinogens are readily autooxidized to their respective colored porphyrins. These oxidations are catalyzed by light

HEME SYNTHESIS

Uroporphyrinogen III is converted to coproporphyrinogen III by decarboxylation of all the acetate groups Coproporphyrinogen III enters the mitochondria where it is converted to protoporphyrinogen III and then to protoporphyrin III, the parent porphyrin of heme

HEME SYNTHESIS

85% of heme synthesis occurs in the bone marrow and majority of the remainder is made in hepatocytes

ALA SYNTHASE
The key regulatory enzyme in the biosynthesis of heme ALA synthase occurs in both hepatic (ALAS 1) and erythroid (ALAS 2) forms Heme acts as a negative regulator of the synthesis of ALAS I

ALA SYNTHASE
Many drugs can result in an increase in ALAS 1 due to heme utilization by cytochrome p450 for their metabolism e.g. Morphine, Phenobarbital ALAS 2 does not undergo feedback regulation by heme

PORPHYRINS VS. PORPHYRINOGENS

Porphyrinogens are colorless, whereas the various porphyrins are all colored The double bonds joining the pyrrole rings in the porphyrins are responsible for their fluorescence Spectrophotometry is used to test for porphyrins and their precursors (for diagnosis of porphyrias)

PORPHYRIAS
A group of disorders due to abnormalities in the pathway of biosynthesis of heme Genetic or acquired

PORPHYRIAS
If the enzyme lesion occurs early in the pathway prior to formation of porphyrinogens, ALA and PBG accumulates in body tissues causing abdominal pain and neuropsychiatric symptoms Later blocks cause photosensitivity

PORPHYRIAS
Treatment is symptomatic Ingestion of large amounts of carbohydrates or administration of hematin may repress ALAS 1 resulting in diminished production of harmful heme precursors Patients experiencing photosensitivity may benefit from administration of beta-carotene, aside from administering sunscreens

Biochemical causes of the major signs and symptoms of the porphyrias

HEME CATABOLISM
Under physiologic conditions, 1-2 x 108 erythrocytes are destroyed per hour When hemoglobin is destroyed, globin is degraded into its constituent amino acids, which are then reused Iron is also reused The iron-free porphyrin is also degraded in the reticuloendothelial cells of the liver, spleen, and bone marrow

HEME CATABOLISM
In birds and amphibians the final product is the green biliverdin IX In mammals, biliverdin reductase reduces the methenyl bridge between pyrrole III and IV to a methylene group to produce a yellow pigment, bilirubin The chemical conversion of heme to bilirubin by reticuloendothelial cells can be observed in vivo as the purple color of the heme in a hematoma is slowly converted to the yellow pigment of bilirubin.

HEME CATABOLISM
Bilirubin formed in peripheral tissues is transported to the liver by plasma albumin The further metabolism of bilirubin occurs primarily in the liver.

BILIRUBIN CATABOLISM
Divided into three processes: 1. Uptake of bilirubin by liver parenchymal cells 2. Conjugation of bilirubin with glucuronate in the endoplasmic reticulum 3. Secretion of conjugated bilirubin into the bile.

BILIRUBIN CATABOLISM
Bilirubin is nonpolar, so hepatocytes conjugate it to make it water-soluble by adding glucuronic acid; then, it is excreted in the bile The conjugation of bilirubin is catalyzed by a glucuronosyltransferase The enzyme is mainly located in the endoplasmic reticulum, uses UDP-glucuronic acid as the glucuronosyl donor, and is referred to as bilirubinUGT

Structure of bilirubin diglucuronide (conjugated, "direct-reacting" bilirubin)

Glucuronic acid is attached via ester linkage to the two propionic acid groups of bilirubin to form an acylglucuronide

Conjugation of bilirubin with glucuronic acid

The glucuronate donor, UDP-glucuronic acid, is formed from UDP-glucose The UDP-glucuronosyltransferase is also called bilirubinUGT.

BILIRUBIN CATABOLISM
Most bilirubin is excreted in the form of bilirubin diglucuronide Phenobarbital induces bilirubin-UGT activity, which makes it effective in the treatment of unconjugated hyperbilirubinemia in infants

BILIRUBIN SECRETION INTO BILE

Occurs by active transport Protein involved is MRP-2 (Multidrugresistance-like protein 2) or MOAT (Multispecific organic ion transporter) located in the plasma membrane of the bile canalicular membrane

BILIRUBIN SECRETION INTO BILE

As the conjugated bilirubin reaches the terminal ileum and large intestine, the glucuronides are removed by glucuronidases, which are bacterial enzymes The pigment is subsequently reduced to urobilinogen, a colorless compound

BILIRUBIN SECRETION INTO BILE

Most of the colorless urobilinogens are oxidized to colored urobilins in the colon and are excreted in feces A small fraction of urobilinogens is reabsorbed and reexcreted through the liver (enterohepatic urobilinogen cycle )

 In the lower small intestine and colon, bacteria remove glucuronic acid residues and reduce bilirubin to the colorless urobilinogen and stercobilinogen. Exposure to air oxidizes these to urobilin and stercobilin, respectively, (red-orange pigments that contribute to the normal color of stool and urine).

 Urobilinogen is excreted mostly in the feces, but a small fraction is absorbed from the colon, enters the portal circulation, is removed by the liver, and is secreted into bile. That which is not removed from the portal blood by the liver enters the systemic circulation and is excreted by the kidneys.

 Urobilinogen excretion in urine normally amounts to 1-4 mg per 24 hours, as opposed to the 40-280 mg excreted in feces.

 Lack of urobilinogen in the urine and feces indicates biliary obstruction Stools are whitish ("clay-colored") owing to the absence of bile pigment. Urinary and fecal urobilinogen excretion increases in hemolytic anemia.

HYPERBILIRUBINEMIA AND JAUNDICE

Hyperbilirubinemia occurs when bilirubin in the blood exceeds 1 mg/dL (17.1 mol/L) May be due to excess bilirubin production or liver failure Obstruction of the excretory ducts of the liver also prevents bilirubin excretion and causes hyperbilirubinemia When bilirubin reaches 2-2.5 mg/dL in blood, it diffuses into tissues causing jaundice or icterisia

TYPES OF HYPERBILIRUBINEMIA
1. Retention hyperbilirubinemia due to overproduction of bilirubin (Unconjugated bilirubin) 2. Regurgitation hyperbilirubinemia due to the reflux into the bloodstream secondary to biliary obstruction (Conjugated bilirubin)

 Because of its hydrophobicity, only unconjugated bilirubin can cross the bloodbrain barrier into the central nervous system Thus, encephalopathy due to hyperbilirubinemia (kernicterus) can occur only in connection with unconjugated bilirubin, as found in retention hyperbilirubinemia.

 On the other hand, because of its watersolubility, only conjugated bilirubin can appear in urine Choluric jaundice (choluria is the presence of bile pigments in the urine) occurs only in regurgitation hyperbilirubinemia Acholuric jaundice occurs only in the presence of an excess of unconjugated bilirubin

EHRLICH TEST FOR BILIRUBIN

Based on the coupling of diazotized sulfanilic acid and bilirubin to produce a reddish-purple azo compound A reaction without methanol being added means that conjugated bilirubin is present; with methanol it is unconjugated bilirubin

UNCONJUGATED HYPERBILIRUBINEMIA
1. 2. Hemolytic anemia Neonatal Physiologic jaundice A transient condition Most common cause of unconjugated hyperbilirubinemia Due to accelerated hemolysis with an immature liver

UNCONJUGATED HYPERBILIRUBINEMIA
3. Crigler-Najjar syndrome type I Congenital nonhemolytic jaundice Autosomal recessive Due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues 4. Crigler-Najjar syndrome type II Like type I but more benign

UNCONJUGATED HYPERBILIRUBINEMIA
5. Gilbert syndrome Also has mutations in genes encoding bilirubin-UGT but retains 30% of enzyme activity 6. Toxic hyperbilirubinemias Chloroform, carbon tetrachloride, acetaminophen, viral hepatitis, cirrhosis, Amanita mushroom

CONJUGATED HYPERBILIRUBINEMIA
1. 2. Biliary Obstruction Regurgitation into hepatic veins and lymphatics Conjugated bili in blood and urine Dubin-Johnson syndrome Benign autosomal recessive Mutation in the gene encoding MRP-2 for the secretion of conjugated bili into bile 3. Rotor syndrome Chronic conjugated hyperbilirubinemia and normal liver

Laboratory Results in Normal Patients and Patients with Three Different Causes of Jaundice.
Serum Bilirubin
Normal Direct: 0.10.4 mg/dL
Indirect: 0.20.7 mg/dL Hemolytic anemia Hepatitis Indirect Direct and indirect Direct Increased Decreased if microobstruction is present Absent Absent Present if microobstruction occurs Present Increased Decreased

Urine Urobilinogen
04 mg/24 h

Urine Bilirubin
Absent

Fecal Urobilinogen
40280 mg/24 h

Obstructive jaundice1

Trace to absent